Determining the Condition of a Wound

Abstract

A product for monitoring the condition of a wound comprises: (a) a sample application zone; (b) a reaction zone downstream of the sample application zone comprising protease-sensitive polymers; (c) coloured particles; and (d) a viewing zone downstream of the sample application zone and reaction zone. The sample application zone transmits fluid towards the viewing zone. Cleavage of the polymers by protease activity present in applied wound fluid results in carriage of the coloured particles with the wound fluid to the viewing zone thereby providing a visual indication of protease activity in the viewing zone. A product for monitoring the condition of a wound comprises a matrix that absorbs wound fluid. The matrix comprises cross-linked and protease-sensitive polymers forming a reaction zone on/in the matrix, and, coloured particles. The arrangement of the polymers and coloured particles is such that cleavage of the polymers by protease activity present in the wound fluid results in transport of the coloured particles along/through the matrix to provide a visual indication of protease activity in the wound fluid. Companion products, uses, kits and methods of monitoring the condition of a wound are also provided.

Claims

1.-33. (canceled)

34. A product for monitoring the condition of a wound comprising: a. a sample application zone to which wound fluid is added b. a reaction zone downstream of the sample application zone comprising protease-sensitive polymers; c. a zone comprising coloured particles downstream of the sample application zone; d. a viewing zone downstream of the sample application zone, reaction zone and zone comprising coloured particles; wherein the sample application zone transmits fluid towards the viewing zone and wherein cleavage of the polymers by protease activity present in the wound fluid results in carriage of the coloured particles with the wound fluid to the viewing zone thereby providing a visual indication of protease activity in the viewing zone, wherein: (i) the coloured particles are entrapped within the polymers and cleavage of the polymers by protease activity present in the wound fluid results in release of the coloured particles to the viewing zone thereby providing a visual indication of protease activity in the wound fluid via the viewing zone; or (ii) the reaction zone forms a barrier that prevents wound fluid reaching the viewing zone and wherein cleavage of the polymers by protease activity present in the wound fluid, optionally above a threshold level, disrupts the barrier and results in carriage of the coloured particles to the viewing zone thereby providing a visual indication of protease activity in the wound fluid via the viewing zone.

35. The product of claim 34 wherein the polymers are cross-linked.

36. The product of claim 34 (ii) wherein the zone comprising coloured particles is downstream of the reaction zone.

37. The product of claim 34 wherein the product comprises a visual symbol that, prior to exposure to wound fluid and in the absence of protease activity, is masked by the coloured particles and wherein the visual indication of protease activity in the wound fluid comprises revelation of the visual symbol.

38. The product of claim 37 which defines a second viewing zone positioned above the zone comprising coloured particles prior to exposure to wound fluid and in the absence of protease activity.

39. The product of claim 34 wherein: (i) the viewing zone comprises capture molecules to capture coloured particles in the viewing zone; (ii) the product further comprises a barrier at the downstream end of the viewing zone so that coloured particles accumulate in the first viewing zone; and/or (iii) the sample application zone and reaction zone at least partially overlap.

40. The product of claim 39 wherein the capture molecules comprise antibodies that bind specifically to the coloured particles.

41. A method of monitoring the condition of a wound comprising: a. applying a sample of wound fluid to the sample application zone of a product as claimed in claim 34 b. detecting the visual indication of protease activity provided by the coloured particles in the viewing zone.

42. The method of claim 41 wherein (i) the measured coloured particles in the viewing zone provide a quantitation of the protease activity in the wound fluid; and/or (ii) the product further comprises a second viewing zone and loss of coloured particles in the second viewing zone is compared with gain in coloured particles in the first zone.

43. A product for monitoring the condition of a wound comprising a matrix that absorbs wound fluid, the matrix comprising: a. cross-linked and protease-sensitive polymers forming a reaction zone on/in the matrix b. coloured particles; wherein the arrangement of the polymers and coloured particles is such that cleavage of the polymers by protease activity present in the wound fluid results in transport of the coloured particles along/through the matrix to provide a visual indication of protease activity in the wound fluid; wherein the matrix comprises a viewing zone that, prior to exposure to wound fluid and in the absence of protease activity, does not contain coloured particles and wherein cleavage of the polymers by protease activity present in the wound fluid results in release of the coloured particles along/through the matrix providing a visual indication of protease activity in the viewing zone, wherein: (i) the coloured particles are entrapped within the polymers and cleavage of the polymers by protease activity present in the wound fluid results in release of the coloured particles along/through the matrix to provide a visual indication of protease activity in the wound fluid; or (ii) the cross-linked and protease-sensitive polymers form a barrier that, in the absence of protease activity, optionally above a threshold level, in the wound fluid, prevents the wound fluid from coming into contact with the coloured particles and wherein cleavage of the polymers by protease activity present in the wound fluid, optionally above a threshold level, disrupts the barrier and results in flow of the coloured particles along/through the matrix to provide a visual indication of protease activity in the wound fluid in the viewing zone.

44. The product of claim 43 wherein (i) the visual indication comprises dispersal of the coloured particles; (ii) the matrix visible in the viewing zone comprises capture molecules to capture coloured particles; and/or (iii) the matrix comprises a barrier aligned with the (downstream) end of the viewing zone so that coloured particles accumulate in the viewing zone.

45. The product of claim 43 (i) wherein the matrix comprises a visual symbol that, prior to exposure to wound fluid and in the absence of protease activity, is masked by the coloured particles entrapped within the polymers and wherein the visual indication of protease activity in the wound fluid comprises revelation of the visual symbol as the polymers are cleaved and the coloured particles released.

46. The product of claim 43 wherein: (i) the coloured particles comprise polystyrene microparticles; (ii) the polymers comprise collagen polymers; (iii) the polymers comprise gelatin polymers; and/or (iv) the protease is a serine protease, cysteine protease, aspartic protease, threonine protease and/or glutamic protease.

47. The product of claim 43 (ii) wherein the matrix comprises a visual symbol that, prior to exposure to wound fluid and in the absence of protease activity, is masked by the coloured particles and wherein the visual indication of protease activity in the wound fluid comprises revelation of the visual symbol as the polymers are cleaved, the barrier disrupted and the coloured particles flow along/through the matrix.

48. The product of claim 43 wherein: (i) the cross-links comprise methacrylate or derivatives thereof; or (ii) the cross-links are derived from glutaraldehyde or derivatives thereof.

49. A method of monitoring the condition of a wound comprising: a. applying a sample of wound fluid to a product as claimed in claim 43 b. detecting the visual indication of protease activity provided by the coloured particles.

50. The method of claim 49 wherein the coloured particles are measured in a region of the matrix to provide a quantitation of the protease activity in the wound fluid, optionally wherein: (i) the region comprises a viewing zone; and/or (ii) there are two viewing zones and loss of coloured particles in one zone is compared with gain in coloured particles in a second zone.

51. A kit for making a product as claimed in claim 43 comprising: a. cross-linked and protease-sensitive polymers b. coloured particles.

52. The kit of claim 51, further comprising a matrix, optionally further comprising a housing to contain the matrix, optionally wherein the housing comprises one or more, optionally two, viewing windows for viewing the coloured particles.

53. A kit of parts comprising: (i) a product comprising a sample application zone, reaction zone (absent of (optionally cross-linked) protease-sensitive polymers) and a viewing zone; (ii) protease-sensitive polymers; (iii) coloured particles; and (iv) optionally one or more cross-linking agents.

54. The product of claim 34 wherein: (i) the coloured particles comprise polystyrene microparticles; (ii) the polymers comprise collagen polymers; (iii) the polymers comprise gelatin polymers; and/or (iv) the protease is a serine protease, cysteine protease, aspartic protease, threonine protease and/or glutamic protease.

55. The product of claim 35 wherein: (i) the cross-links comprise methacrylate or derivatives thereof; or (ii) the cross-links are derived from glutaraldehyde or derivatives thereof.

Description

DESCRIPTION OF THE FIGURES

[0338] FIG. 1A is a schematic illustrating one embodiment of the invention wherein a product as described herein is placed in contact with a wound underneath a wound dressing.

[0339] FIG. 1B is a schematic illustrating the generation of a visual indication by a product as described herein following absorption of wound fluid and cleavage of the cross-linked protease-sensitive polymers by protease activity present in the wound fluid.

[0340] FIG. 2 is a schematic illustrating a lateral-flow based embodiment of a product as described herein.

[0341] FIG. 3 is a schematic illustrating a further lateral-flow based embodiment of a product as described herein.

[0342] FIG. 4 shows the NMR spectra of unmodified gelatin prior to methacrylate modification.

[0343] FIG. 5 shows the NMR spectra of fully methacrylate modified gelatin (GELMA).

[0344] FIG. 6 shows a top-view (A) and exploded side-view (B) of an exemplary product according to the invention, preferably for in situ use in a wound.

[0345] FIG. 7 demonstrates a product as described herein in use (time=Ohr).

[0346] FIG. 8 shows the results using the product shown in FIG. 7 in the presence and absence of papain after 24, 48 and 72 hr.

[0347] FIG. 9 demonstrates a further product as described herein in use (time=Ohr).

[0348] FIG. 10 shows the results using the product shown in FIG. 9 in the presence and absence of matrix metalloproteinase 9 (MMP9) and human neutrophil elastase (HNE) after 24 and 48 hr.

[0349] FIG. 11 shows the results using the product shown in FIG. 9 in the presence and absence of matrix metalloproteinase 9 (MMP9) and human neutrophil elastase (HNE) after 120 and 144 hr.

DETAILED DESCRIPTION OF THE INVENTION

[0350] The invention will now be described, without limitation but solely to aid understanding of the invention, by reference to the Figures.

[0351] A product (1) as described herein is shown schematically in FIG. 1A. The product (1) comprises, consists essentially of or consists of a matrix (2), preferably biologically inert for in situ applications, which can absorb wound fluid and coloured particles entrapped within cross-linked and protease-sensitive polymers (3), in this case coloured polystyrene microspheres (PSM) entrapped within cross-linked gelatin, on or in the matrix (2). The coloured particles entrapped within the cross-linked gelatin (3) may be dried into, or conjugated to, the matrix (2) and, in the case of suitably coloured PSM, make the whole or a substantial part of the matrix appear black in colour, forming a test unit or reaction zone. The coloured particles entrapped within the cross-linked gelatin (3) are used to measure protease (gelatinase) activity in fluid released by a wound. The cross-linked gelatin (3) is degraded by gelatinases present (optionally at or above a threshold concentration) in the wound fluid.

[0352] When the product (1) is placed in contact with the wound (4), which may be a chronic wound, on a subject (5) underneath a wound dressing (6), the matrix (2) absorbs wound fluid. If the wound fluid comprises sufficient gelatinase activity, the cross-linked gelatin (3) on or in the matrix (2) is degraded into fragments, in particular if the gelatinase activity is present at or above a threshold level. Once degraded, the PSM are no longer entrapped and are free to disperse through/along the matrix. In this case, as shown in FIG. 1B, dispersal of the PSM through/along the matrix removes the black colouration that was attributed to the reaction zone of the matrix by the PSM prior to exposure to wound fluid and reveals a visual symbol printed on the product such as a cross (7). The revelation of the visual symbol indicates the presence of aberrant protease activity in the wound.

[0353] While the cross (7) provides a positive test result, and is therefore advantageous, it is not essential. Instead dissipation of the colouration can be used as an outcome of the test without revealing a further symbol.

[0354] FIG. 2 is a schematic illustrating a lateral-flow based embodiment of a product as described herein. A sample application zone is shown (21) to which is applied wound fluid (represented by the straight grey arrows (22)). In this particular case, the sample application zone is a flexible wick which acts as a swab for collecting the wound fluid (22).

[0355] This wick (21) is fluidly connectable with solid support (23) which in this case is a porous test strip in which fluids flow by capillary action (analogous to that of a lateral flow immunoassay). Methacrylate cross-linked gelatin (GELMA) in which coloured PSM are entrapped form a reaction zone (24) on the solid support. Wound fluid is transmitted to the reaction zone (24) via capillary action. Protease activity present in the wound fluid cleaves the GELMA and releases the PSM. The shear stress of the moving fluid entrains the released PSM and these are carried forward with the fluid into a viewing zone (25) thereby providing a visual indication of protease activity in the wound fluid.

[0356] In alternative embodiments, the gelatin may be cross-linked using glutaraldehyde or a derivative thereof instead of methacrylate. Prior to exposure to wound fluid, the gelatin, coloured PSM and gluteraldehyde may be mixed together and applied as a mixture to form the reaction zone (24) on the solid support. Still prior to exposure to wound fluid, the mixture may be dried to promote cross-link formation and to fix the gluteraldehyde cross-linked gelatin, in which the coloured PSM are thus entrapped, to the solid support (23) thereby forming the reaction zone (24).

[0357] In further embodiments, the product, comprising at least the solid support (23) and optionally also the wick (21) may be housed in a casing with a viewing window downstream (in the direction of fluid travel) of the reaction zone (24). In such embodiments, the viewing window defines the viewing zone (25). Thus, PSM released following cleavage of the GELMA or gluteraldehyde cross-linked gelatin by protease activity in the wound fluid can be observed in the viewing window. Optionally, a second viewing window can be positioned above the reaction zone (24). Thus, loss of colour can be observed through this additional viewing window should PSM be released due to protease activity present in the wound fluid.

[0358] In yet further embodiments, the viewing zone (25) may comprise, consist essentially of or consist of capture molecules capable of specifically binding the PSM. Thus, released PSM are arrested in position and concentrated to assist observation and potentially quantitation.

[0359] The capture molecules may comprise, consist essentially of or consist of antibodies which specifically recognise antigens bound onto the surface of the PSM or it could comprise, consist essentially of or consist of some other molecular species to which the PSM can be made to bind, including charged molecules to bring about ion-exchange interactions.

[0360] In further embodiments, the device can be coupled with a source of matched buffer solution such as phosphate buffered saline pH 7.2 (with surfactant to prevent unwanted adhesion of PSM to the test strip) to act as a chase fluid in order to carry the wound fluid through the pores of the test strip and maximise the extraction of PSM from the matrix (when proteolysis has started) and efficiently transport them to the viewing zone (24).

[0361] FIG. 3 is a schematic illustrating a further lateral-flow based embodiment of a product as described herein.

[0362] The product comprises, consists essentially of or consists of a solid support (30) comprising, consisting essentially of or consisting of a sample application zone comprising, consisting essentially of or consisting of an absorbent material (31), a barrier (reaction zone) formed from protease-sensitive polymer molecules, in this case gelatin (32), a zone comprising, consisting essentially of or consisting of coloured particles (34), in this case Monastral blue dye molecules (MBDM), and a viewing zone (35). Optionally, the product also comprises etchings (33) to guide and concentrate any fluid along/through the matrix.

[0363] In operation, a sample of wound fluid is added to the sample application zone (31) and travels via capillary action in the direction indicated by the large arrow (shown in FIG. 3 parallel to the product schematic) until it reaches the gelatin barrier (32). In the absence of protease activity in the wound fluid, the gelatin plug remains intact and prevents the wound fluid from passing beyond it. Thus, the MBDM remain dried to the matrix and are not transmitted toward and into the viewing zone. Consequently, no visual indication of protease activity is provided in the viewing zone. However, in the presence of protease (gelatinase) activity in the wound fluid, the gelatin plug is cleaved and wound fluid can now travel via capillary action toward and into the viewing zone. In so doing, the wound fluid carries with it MBDM into the viewing zone thereby providing a visual indication of protease activity in the wound fluid.

[0364] In further embodiments, the solid support (30) may be housed in a casing with a viewing window downstream (in the direction of fluid travel) of the zone comprising, consisting essentially of or consisting of coloured particles (34), and which encompasses, and may define, the viewing zone (35). Thus, coloured particles (e.g. MBDM) released following cleavage and disruption of the gelatin barrier by protease activity in the wound fluid can be observed in the viewing window. Optionally, a second viewing window can be positioned above the zone comprising, consisting essentially of or consisting of coloured particles (34). Thus, loss of colour can be observed through this additional viewing window should MBDM be released due to protease activity present in the wound fluid.

EXPERIMENTAL SECTION

Example 1: Production of GELMA from Gelatin and Methacrylate

[0365] Gelatin was modified by end-capping with methacrylic anhydride, affording methacrylamide terminated gelatin, termed herein GELMA (general scheme shown below).

##STR00001##

[0366] There are many references in the literature which use a variety of different methods and conditions to produce this material but the method used by Bae Hoon Lee et. al (RSC adv., 2015, 5, 106094) was adopted for this purpose, as follows.

[0367] Type A gelatin, obtained by acid treatment at pH1-2 (an isoelectric point of pH7-9) was used. In order to get efficient substitution (endcapping) a high excess of methacrylic anhydride was needed. Since the by-product from the reaction is methacrylic acid the pH of the reaction was gradually lowered to below the iso-electric point range, resulting in reduced endcapping. To circumvent this, Bae Hoon Lee et. al. used a carbonate buffer to keep the reaction mixture between pH 7 & 9 and the alternate addition of methacrylic anhydride and sodium hydroxide for pH control. This method allowed for efficient endcapping with the least amount of side reactions (termination of hydroxyl groups with methacrylic ester). It also reduced the amount of reagents used and minimised the amount of inorganic salts to be removed in the purification step. The reaction product was purified by tangential flow filtration (TFF), although simple dialysis can also be used.

Detailed Method

[0368] Gelatin type A [175 bloom] (10 g) and 100 ml of carbonate buffer 0.1M (100 ml) [3.18 g NaCO3, 5.86 g NaHCO3] were mixed in a round bottomed flask fitted with an overhead stirrer, temperature probe and pH meter. The mixture was heated to 60 C. to dissolve the solid. The reaction temperature was then reduced to 50 C. and the pH checked and adjusted to 9.0.

[0369] Methacrylic acid (167 l) was added dropwise to this solution causing the pH to drop to 8.5 and the reaction mixture was stirred for 25 mins. After this, the pH was then adjusted back to pH 9.0 with 20% NaOH solution and stirred for a further 5 mins.

[0370] Further rounds of methacrylic acid and pH adjustment were repeated a further 5 times until a total of 1 mL of methacrylic acid had been added during a total of 3 hrs reaction time.

[0371] Finally, the pH was adjusted to 7.4 with 50% acetic acid. The resultant mixture was filtered through 0.2 micron PTFE filter membrane into a tangential flow filtration reservoir. The mixture was then purified by dia-filtering against water (water for irrigation) using a 3 kD midi-Kros MPEs TFF membrane with a transmembrane pressure of 15. A total of 6 L was dia-filtered to obtain a permeate conductivity <10 S. The resulting pale yellow solution was transferred to a Florentine flask and freeze dried for 2 days. This afforded a white polystyrene-like material 8.7 g, ready for use in preparing a product as described herein. The degree of methacrylate substitution in the product was determined by proton NMR. The results are shown in FIGS. 4 and 5. Note the loss or depletion of peaks from FIG. 4 (e.g. the lysine methylene peak at 3 ppm) and the appearance of new peaks in FIG. 5 (e.g. at 4.8 ppm, 5.7 and 5.9) indicating the incorporation of methacrylate groups in the GELMA product. These results confirm the reaction to have progressed well with the loss of peak at 2.95-3.1 ppm.

Elemental Analysis. (CHN and Na)

[0372]

TABLE-US-00001 Element Carbon Hydrogen Nitrogen Sodium % Found 45.88 6.42 16.21 0.71

[0373] These data show there is still some sodium present but this did not cause a problem.

Discussion

[0374] This method had been followed twice on a 2 g pilot scale. One reaction product was purified by dialysis (12-14 KD cut off), the other by TFF (3 KDa cut off). Both materials performed equally well for manufacture of a product as described herein. Due to the ease and efficiency of the TFF purification it was decided to use this method to purify the larger 10 g batch. The method was reproducible, simple and efficient.

Conclusion

[0375] The method used to generate gelatin methacrylate (GELMA) is fit for this purpose. If required, the skilled person could further optimise the formulations by known methods and approaches, such as constant pH control with automated base addition. Scale-up for commercial application can be readily achieved by known methods and procedures.

Example 2: Manufacture of a Product According to the Invention

[0376] The manufacturing process includes all or some of the following operations, the details of which can be optimised in accordance with the needs of user and the constraints of the manufacturing plant.

TABLE-US-00002 Step Task 1 Coloured reagent preparation 2 Absorbent material 3 Permanent + indicator printing 4 Deposition of coloured reagent 5 Fixation of coloured reagent (achieved by cooling to solidify, and exposure to UV) 6 Drying of the coloured reagent 7 Application of top cover 8 Conversion and packaging 9 Sterilisation

[0377] A particular non-limiting format, which has been found to be useful as a practical and readily manufactured product is as follows: [0378] A disk of absorbent medical material made from a medical foam measuring 2.5 mm in depth and with a diameter of 15 mm diameter. [0379] A + symbol is printed with permanent ink on the top side of the disk in a central position. The printed + measures 4 mm across. [0380] The + is covered (therefore hidden from view) by a coloured reagent comprising, consisting essentially of or consisting of cross-linked methacrylate modified gelatin (GELMA) [0381] The product has an elastic, thin, transparent covering material adhered to the top surface. The preferred covering is a medical grade polyurethane. [0382] The individual 15 mm disks are packaged as single unit, and sterilised.

[0383] A schematic of such a product is shown in FIG. 6. FIG. 6A shows a top-view of the product. The disc marked with a permanent cross is covered with and obscured by the application of the coloured reagent comprising, consisting essentially of or consisting of cross-linked methacrylate modified gelatin (GELMA). FIG. 6B shows an exploded side-view of the product comprising, consisting essentially of or consisting of a polyurethane film (61), the coloured reagent comprising, consisting essentially of or consisting of cross-linked methacrylate modified gelatin (GELMA) (62) and an absorbent matrix (63).

Example 3: ManufactureWet Deposition of the GELMA Reagent and Assembly

[0384] Stock materials required for product: [0385] Absorbent material roll stock [0386] Polyurethane roll stock (with adhesive already applied) [0387] Modified gelatin (GELMA) [0388] Dyed polystyrene microparticles [0389] Permanent ink [0390] Moisture impermeable packaging (likely to be laminated foil)

[0391] The following steps constitute the manufacturing process:

TABLE-US-00003 Step Task Description 1 Coloured Modified gelatin GELMA is prepared, simply by dissolving into reagent water at elevated temperature. Coloured polystyrene preparation microparticles are added to the mix. The mix is kept at 30 C. or above to prevent solidification. 2 Absorbent Roll stock of absorbent material unwound onto assembly material equipment 3 Permanent + A printing station applies the + symbol. The ink needs to be mark printing fixed in place before the application of the coloured reagent. By heat drying for solvent based ink By UV for UV activated ink 4 Deposition of A fixed volume (5 microlitres) of the GELMA and polystyrene coloured microparticle mix is applied to cover the printed permanent + reagent symbol. The storage vessel and deposition lines/nozzle maintained at a temperature of 30 C. or above to prevent solidification of the coloured reagent and therefore blocking of the equipment. This can be achieved by keeping the temperature of the room at a minimum of 30 C. 5 Fixation of The coloured reagent stays in place after deposition due to the coloured viscosity of the solution. reagent The deposited coloured reagent is then fixed with high intensity (achieved by UV. Exposure time is 15-30 seconds. cooling to solidify, and exposure to UV) 6 Drying of the Due to the small volume of the deposited reagent, the drying is coloured a quick process, accelerated by passing through a heated air reagent tunnel. 7 Application of A medical polyurethane (PU) cover is applied to the absorbent top cover product. This can be achieved by the use of a pre-coated PU, or By applying adhesive to the absorbent material then apply an un-coated PU 8 Conversion 15 mm disks are cut from the finished laminated roll-stock, and and packaging individually packaged. 9 Sterilisation The final product will be sterilised. This can be achieved by gamma or e-beam irradiation, or by ethylene oxide or dry heat (e.g. in sealed pouches in an autoclave at 121 C.) NOTES: Step 4: The coloured reagent needs to be kept at an elevated temperature during storage and deposition. This is to prevent the reagent from solidifying. The volume to be deposited is very small (5 microlitres per disk). The viscosity of the warmed modified gelatin is not high, although it is higher than water. If necessary, the viscosity can be enhanced by adding a thickening agent (e.g. carboxy methyl cellulose or just more unmodified gelatin). The coloured reagent should not be absorbed into the absorbent material, otherwise the indicator matrix will not become uniformly cross-linked and the depth of the matrix it forms will be compromised. This has an effect on the choice of the absorbent material and/or the properties of the pre-cross-linked GELMA reagent. The deposition of the coloured reagent needs to be aligned with the printed + symbol. The coloured reagent can be circular in presentation, as long as it covers (obscures) all of the pre-printed + symbol. Registration is therefore important. Step 5: A UV lamp is used to fix the coloured reagent in place. The exposure time is 15-30 seconds, depending on the power output of the lamp. So far, lamps which deliver ~300 nm and ~380 nm UV lamps, at energy levels ranging from 1-100 mW/cm.sup.2 have been found to be effective. Step 6: The fixed coloured reagent is dried before packaging. A hot air tunnel may be used to dry the coloured, matrix protease. Other options may be considered.

Problems Encountered and Overcome by the Exemplified Product for In Situ Use

Problems Encountered:

[0392] 1) Modifying the properties of dried gelatin to make it more suitable for tests in which the matrix is exposed to wound fluid for extended periods. Gelatin (denatured collagen) in its normal state (as purchased or derived from collagen) can be dried to form suitable matrix structures but it is gradually dissolved by aqueous fluids containing dissolved proteins, such as wound fluids at typical wound temperatures, even in the absence of protease. On the other hand, excessively modified gelatin can become too resistant to protease cleavage. Although un-modified gelatin can work under certain conditions, the cross-linked version is more robust and capable of working over longer time periods. [0393] 2) In manufacturing, the deposited gelatin solution must be very quickly fixed in place, such that it becomes firmly adhered to the carrier material, otherwise it will become smeared or dislodged. Alternatively very slow, uneconomic processing would have to be used. [0394] 3) Dye-modified gelatin may not form a sufficiently intense colour when deposited on a suitable carrier surface in a depth sufficiently thin to allow destruction by clinically relevant levels of protease. [0395] 4) The simplicity of operation desired for this application does not allow for complex processes such as immunoassays or other procedures that require more than a single operational step. [0396] 5) Because the device must be in physical contact with the wound, it is not possible to use reactive ingredients which have not been approved for use in wounds or do not have a history of use in wounds. [0397] 6) Some aspects of operation planned for the device require that it can be left in-situ on the surface of the wound, under a dressing for at least one day and potentially for several days.

TABLE-US-00004 Problem to be overcome How the problem is overcome Comments 1 Controlled modification of lysine epsilon This approach prevents amino groups in the gelatin molecules with premature solubilisation of polymerisable functional groups such as dried gelatin by simple methacrylate (to form GELMA), which allows dissolution in aqueous fluid. subsequent controlled cross-linking. It requires protein chain Alternatively, direct cross-linkers such as cleavage before the matrix glutaraldehyde may be used if carefully controlled. can be dissolved. 2 During manufacture, with methacrylate This is highly compatible modification, the GELMA cross-linking can with production line be delayed until the liquid matrix is processing and yields an dispensed onto the carrier, whereupon ideal indicator matrix irradiation with UV light causes an immediate, robust cross-linking to occur. 3 Instead of using soluble dyes that associate Very dense coloration of the (reversibly) with the gelatin molecules, or are matrix is achieved by the covalently attached to the gelatin, coloured use of CPSM. The process polystyrene microparticles (CPSM) are of CPSM escape and mixed with the GELMA. These become consequent decolourisation firmly enmeshed in the cross-linked gelatin happens easily and without molecular network, but readily escape when agitation of fluid flow, even the gelatin molecules are cleaved by the in completely static proteases. conditions. 4 CPSM entrapment in the cross-linked This is a true single-step GELMA molecular network provides a process and is unlike any coloured matrix which then decolourises other protease test simply by dispersal of the particles. As this encountered by the requires no intervention or sequential inventors. processing, the test takes place without any user involvement or any accessory devices, actions or controlled interactions 5 The only chemical entities which come into All of these ingredients are contact with the wound are gelatin, gelatin known to be basically fragments and methacrylate-amino-acid biocompatible. compounds. Of course, gelatin is a natural substance with a long history of safe use in wounds. Methacrylate componds have long been in use in wounds as ingredients of polymeric hydrogels. Any dye molecules are entrapped within the CPSM, and polystyrene particles are non-toxic substances, varieties of which have been approved for use in wounds. The carrier material is also selected from materials already approved for use in wounds. 6 Because of the basic biocompatibility of the The functioning of the ingredients, the extreme simplicity of the protease activity test is not structure and the assay process, together compromised under the with the very low physical profile (no more dressing. It can be left in than 2.5 mm thick, the test device can be place for less than a day or placed on the wound under any cover for many days. dressing without mutual interference.

[0398] The product can be in the form of a very thin structure with little or no fluid collection capability. It can be presented as an in situ test unit built into a non-woven carrier which is simply wettable by wound fluid. It can sit on the wound surface in this mode, where it can function simply as a low-cost, supremely easy-to-use protease activity indicator, under a dressing if required for variable lengths of time. In its very simplest embodiment, it could even be used without a carrier, presented just as a coloured, cross-linked piece of gelatin film, although this would be less easy to handle and observe. Alternatively, the indicator matrix can be part of a composite unit with dimensions of 15 mm2.5 mm, in which there is a transparent cover, a non-woven carrier layer and a foam sample-collector layer.

Example 4: Demonstration of a Product According to the Invention in Use Based on Methacrylate Cross-Linked Gelatin

Materials Used

[0399] GELMA: modified gelatin with methacrylic anhydride. (Methacrylic anhydride sourced from Sigma-Aldrich (product number 276685). Gelatin sourced from Sigma Aldrich, Porcine Type A, 300 bloom (product number G2500))

[0400] Photo initiator Daracure, 2-hydroxy-4-(2hydroxyethoxy)-2-methylpropiophenone sourced from Sigma Aldrich (product number 410896)

[0401] Polybead Polystyrene Blur Dyed Microsphere (0.5 micron diameter, 2.5% solids) sourced from Polysciences, Inc. (product number 15709)

[0402] Polyurethane film sourced from Coveris Advanced Coatings (Wrexham, UK), (Inspire 2331)

[0403] Polyurethane foam sourced from foam Freudenberg, formerly Polymer Health Technologies; (Ebbw Vale, UK), (free sample)

Method

[0404] A 0.5% w/v photo initiator (PI) solution was prepared by adding 2.5 mg of Daracure powder to 500 l 1 concentration phosphate buffered saline (PBS). The mixture was heated to 60 C. with periodic vortex mixing until all solids had dissolved. After the photo initiator had been completely dissolved, 50 mg of GELMA powder was added to the solution, which was gently mixed to allow the GELMA to dissolve. During this dissolution process, the temperature of the GelMA/PI/PBS solution was reduced to 40 C. with periodic vortex mixing. The temperature of the solution was maintained at 40 C. to prevent the GELMA solidifying to a gel. Blue polystyrene microspheres were finally added at a ratio of 9 parts GELMA/PI/PBS to 1 part micro particle stock solution. The solution was mixed thoroughly to ensure a homogenous solution. The final concentrations within the mixture were: 0.45% photo initiator, 8.1% GELMA and 0.25% micro particles.

[0405] 5 l aliquots of the GELMA/microsphere/photo-initiator mix were taken and deposited centrally onto the adhesive surface of a 30 m transparent polyurethane film (Coveris advanced coatings, Inspire 2331) at the rate of one 5 l spot per 1010 mm square of film. Each drop was gently agitated to disperse the drop of blue mixture into a 4-5 mm diameter blue circle. The polyurethane (PU) film squares were then placed under a broad spectrum UV lamp (Dr. Honle, Germany; power c. 100 mW/cm.sup.2) for 15 seconds to initiate crosslinking and polymerise the GELMA. Finally the polymerised spots on the PU squares were air-dried at ambient temperature for 3 hours.

[0406] After the spots had been dried, each PU square (with its own single blue indicator spot) was affixed via the exposed adhesive onto a similarly sized piece of PU foam (5 mm thick), to create a set of composite squares. Thus, each square consisted of a layer of PU foam, topped with a piece of thin transparent PU film, held in place by a layer of adhesive with a dry, blue spot of cross-linked GELMA held between the two layers. The blue GELMA spot was positioned with its uppermost surface in contact with the adhesive and its lower surface in direct contact with the PU foam. Using a permanent marker pen, a 4 mm (height and width) blue cross was drawn on the upper most surface of each composite square (on the top surface of the transparent PU film layer) such that the cross is positioned above the blue GELMA spot so that no part of the drawn cross extended beyond the outer edge of the blue indicator. With this arrangement, the blue cross was visually obscured by the intensely blue spot beneath it. When this had been completed, the squares were ready to be used as indicators of protease activity.

[0407] To test the responsiveness of the assembled indicators, three test solutions were prepared, each based on a standard papain activation buffer with the following composition: 4.22 mM I-Cysteine HCL, 3.6 mM EDTA, 0.2M NaCl in deionised water, pH 7. Test solution 1 contained 0.1 mg/ml of papain (approx. 1000 unit/g), test solution 2 contained 0.001 mg/ml of papain and test solution 3 contained 0 mg/ml papain. Two of the prepared indicator squares were placed in a petri dish containing solution 1, so that the absorbent PU foam became saturated with the solution. In this situation, the solution was brought into direct contact with the blue spot. Two squares were similarly placed in solution 2 and a further two were placed in solution 3. The squares were photographed before they were placed in contact with the test solutions (i.e. in a dry state) and again when initially wetted with the test solutions, and yet again when 24, 48 and 72 hours at 37 C. had elapsed after first contact with the test solutions.

Results

[0408] The state of the blue GELMA spots at the pre-determined time points of the experiment are shown in FIGS. 7 and 8. In the dry state and on first wetting (i.e. time=0 hr) with any of the test solutions it can be seen that the blue GELMA spots are completely intact (FIG. 7). In this condition the blue cross has not been exposed on any of the squares. At each of the subsequent time points almost complete digestion of the GELMA was observed in the presence of both concentrations of papain (FIG. 8). This is seen by the change in presentation, where the blue coloured spot changes to the blue coloured cross. This is because the gelatin component of the GELMA has been digested by the active protease enzyme, which in turn releases the entrapped and immobilised microspheres. The coloured microspheres then disperse, spreading evenly throughout the foam to revealing the indelible cross on the top of PU. In contrast, the blue GELMA spots exposed to the solution with zero papain (negative control) had remained intact throughout the whole time-course, with no observable signs of structural integrity loss (FIGS. 7 and 8). In these squares, the blue coloured circular spot remained, and the blue cross marked on top of the PU film had not been revealed.

Conclusion

[0409] From these results it is clear that the cross-linked GELMA mixed with blue polystyrene microspheres remains intact when exposed to aqueous solutions in which there is no protease activity. When protease activity is introduced to the sample solution, the GELMA is readily broken down by papain (as a representative protease), so releasing the blue microspheres allowing them to disperse throughout the underlying foam layer. This change in state is indicative of protease activity and can be observed either as simple colour dispersal or as the reveal of a superimposed permanent colour-matched cross (or other symbol).

[0410] This result has also been observed with neutrophil elastase and matrix metalloprotease 9, both of which are expected to be present in infected or inflamed wound fluids, as shown in Example 5.

Example 5: Demonstration of a Product According to the Invention in Use Based on Methacrylate Cross-Linked Gelatin

Materials Used

[0411] GelMA: modified gelatin with methacrylic anhydride. (Methacrylic anhydride sourced from Sigma-Aldrich (product number 276685). Gelatin sourced from Sigma Aldrich, Porcine Type A, 175 bloom (product number G2625))

[0412] Photo initiator Daracure, 2-hydroxy-4-(2hydroxyethoxy)-2-methylpropiophenone sourced from Sigma Aldrich (product number 410896)

[0413] Polybead Polystyrene Blur Dyed Microsphere (0.5 micron diameter, 2.5% solids) sourced from Polysciences, Inc. (product number 15709)

[0414] Orion non-woven fabric (4 osy weight) sourced from Anowo Ltd.

Method

[0415] A 0.5% w/v photo initiator (PI) solution was prepared by adding 2.5 mg of Daracure powder to 500 l 1 concentration phosphate buffered saline (PBS). The mixture was heated to 60 C. with periodic vortex mixing until all solids had dissolved. After the photo initiator had been completely dissolved, 50 mg of GELMA powder was added to the solution, which was gently mixed to allow the GELMA to dissolve. During this dissolution process, the temperature of the GELMA/PI/PBS solution was reduced to 40 C. with periodic vortex mixing. The temperature of the solution was maintained at 40 C. to prevent the GELMA solidifying to a gel. Blue polystyrene microspheres were finally added at a ratio of 9 parts GELMA/PI/PBS to 1 part micro particle stock solution. The solution was mixed thoroughly to ensure a homogenous solution. The final concentrations within the mixture were: 0.45% photo initiator, 8.1% GELMA and 0.25% micro particles.

[0416] 1 l aliquots of the GELMA/microsphere/photo-initiator mix were taken and deposited centrally onto 55 mm squares of the Orion non-woven material. A series of repeat depositions were made. The drop was allowed to diffuse into the Orion pad, before being placed under a broad spectrum UV lamp (Dr. Honle, Germany; power c. 100 mW/cm.sup.2) for 15 seconds to initiate crosslinking and polymerise the GELMA. Finally the polymerised spots on the Orion squares were air-dried at ambient temperature for 3 hours.

[0417] To test the responsiveness of the assembled indicators, a series of test solutions were prepared. Matrix metalloproteinase 9 (MMP9) and human neutrophil elastase (HNE) enzymes were diluted into activation buffer with the following composition: 50 mM Tris buffer, 10 mM calcium chloride dihydrate, 100 mM sodium chloride, 50 M zinc chloride, 0.025% w/w Brij 35, 0.05% sodium azide in deionised water. Each enzyme was diluted in the activation buffer to give the following enzyme concentrations: 0, 0.15, 0.31, 0.62, 1.25, 2.5, 5 and 10 micrograms/ml. Two of the prepared indicator squares for each enzyme dilution were placed in a petri dish, providing a series of material squares in an 82 formation. To each pairing, 25 L of the relevant enzyme dilution was added per square. The squares were photographed before they were placed in contact with the test solutions (i.e. in a dry state) and again when initially wetted with the test solutions, and yet again when 24, 48, 120 and 144 hours at 37 C. had elapsed after first contact with the test solutions.

Results

[0418] The results of the experiment are shown in FIGS. 9-11. In the dry state and on first wetting (i.e. time=0 hr) with any of the test solutions it can be seen that the blue GELMA spots are completely intact (FIG. 9). At each of the subsequent time points, digestion of the GELMA was observed in the presence of both MMP9 and HNE (FIGS. 10 and 11). This is indicated by the dispersal (and attenuation) in colour throughout the Orion non-woven material. This is because the gelatin component of the GELMA has been digested by the active protease enzyme, which in turn releases the entrapped and immobilised microspheres. The coloured microspheres then disperse, spreading evenly throughout the Orion non-woven material. In contrast, the blue GELMA spots exposed to the solution which did not contain MMP9 or HNE (0 g/ml; negative control) remained intact throughout the whole time-course, with no observable signs of loss of structural integrity (FIGS. 9-11). The results therefore demonstrate the visible attenuation/loss of colour when the GELMA is exposed to active MMP9 and HNE protease activity. At 24 hours, the higher concentration of both enzymes digested the GELMA and lost the coloured spot (present at time point=0 hr) with the coloured microspheres dispersing throughout the Orion non-woven material. As the incubation time increased, lower concentrations of active enzyme were able to cleave the GELMA and release the coloured microspheres resulting in colour attenuation/loss (FIGS. 10 and 11). This time course clearly demonstrates the GELMA is digestible by both MMP9 and HNE enzymes. The zero enzyme control spots remained intact throughout the test, confirming the crosslinked GELMA does not diffuse or dissolve at 37 C. over an extended incubation period (144 hr).

Conclusion

[0419] From these results it is clear that the cross-linked GELMA mixed with blue polystyrene microspheres remains intact when exposed to aqueous solutions in which there is no protease activity. When active protease activity is present, the GELMA is readily broken down by MMP9 and HNE (representative of biologically relevant enzymes in a wound), so releasing the blue microspheres allowing them to disperse throughout the underlying absorbent layer. This change in state is indicative of protease activity and can be observed either as simple colour dispersal or, in some embodiments, by the consequent revelation of a symbol (e.g. a cross) superimposed or underneath the initial colour spot.

[0420] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.